Date of Award
Doctor of Philosophy (PhD)
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl" channel is an ATP-binding cassette protein, comprising two transmembrane domains, two nucleotide binding domains (NBD1, NBD2) and a regulatory (R) domain. Channel gating is controlled by R-domain phosphorylation and by A T P binding and/or hydroysis at the NBDs. Exon 13 of the C F T R gene encodes residues 590 to 830, originally ascribed to the R domain. Here, CFTR channels were severed near likely N- or C-terminal boundaries of NBD1 or the R domain. Channel activity, assayed via two-microelectrode voltage clamp monitored successful assembly of pairs of channel segments as the sever point was systematically shifted along the primary sequence. Substantial activity indicated successful assembly; such constructs were further studied in excised patches, by correlating macroscopic and single-channel kinetics. The C-terminus of N B D 1 was found to extend beyond residues 590 and lies between residues 622 and 634, while the N-terminus of N B D 1 lies between residues 432 and 449. In excised patches, channels severed just before (between a.a.s 432 and 433) or after N B D 1 (between residues 633 and 634) displayed the usual hallmark characteristics of wild-type (WT) CFTR: requirement of phosphorylation by protein kinase A (PKA) and exposure to A T P for gating, ability to be locked open by pyrophosphate or AMPPNP, small single-channel conductances, and high apparent affinity of channel opening by although bursts of 1-633 plus 634-1480 channels were -40% shorter than in WT. Channels severed near the R domain's C-terminus, 1-835 plus 837-1480, displayed low, PKA-independent, activity, enhanced apparent affinity for ATP, and destabilized binding site for the locking action of AMPPNP. R-domain-less split channels 1-633 plus 837-1480 were functionally similar to 1-835 plus 837-1480, including enhanced apparent ATP affinity and less tight binding of AMPPNP, but were more active before phosphorylation. Intriguingly, 1-633 plus 837-1480 channels, lacking the R domain, were still stimulated by PKA; and AMPPNP or pyrophosphate still prolonged bursts. The kinetic analysis exploited a new method that rapidly extracts single-channel transition rate constants from multichannel patch-clamp recordings, using a simultaneous maximum-likelihood fit to the dwell-time distributions for all conductance levels (after successfully testing it on simulated current traces).
Csanady, Laszlo, "Structure-Function Studies on Human Epithelial Cystic Fibrosis Chloride Channels Expressed in Xenopus Laevis Oocytes" (2000). Student Theses and Dissertations. 320.